La mer 55 : 25Ȃ35, 2017Société franco-japonaise dʼocéanographie, Tokyo

Morphogenesis and growth in the early life stages ofSargassum oligocystum Montagne from fertilized eggs to

juveniles examined in culture

Thidarat NOIRAKSAR1)＊, Vipoosit MANTHACHITRA2), Hisao OGAWA3),Khanjanapaj LEWMANOMONT4) and Ken-ichi HAYASHIZAKI5)

Abstract: Species of Sargassum are widely distributed along the coasts of Thailand. Sargassumoligocystum Montagne is a dominant species consisting of Sargassum beds, playing an importantecological role in a marine ecosystem along the east coast of the Gulf of Thailand. However, thereis little information available on the early life stages of S. oligocystum . To fill the gap in thisecological knowledge, fertilized eggs obtained from the receptacles of wild matured individualswere cultured and morphogenesis in the early life stages of S. oligocystum due to theirdevelopment was observed through laboratory culture. A fertilized egg divided transversely intoone large cell and one small cell. The latter gradually induced rhizoidal cells after several divisionsand many rhizoidal cells came out at the basal part of germling in 3 day culture. Finally, theybecame the holdfast of germling. In the large cell, cell divisions occurred and apical part came outin 1 day culture. It developed into the first cauline leaf in 7 day culture and the fourth cauline leafwas appeared in 30 day culture, which were lanceolate. Cauline leaves were lanceolate to spatulatein 60 day culture and broad spatulate in 90 day culture. Three-month-old juveniles of S.oligocystum were cultured in two 500 L fiberglass tanks set outdoor under a roof with translucentwindows, and one was filled with seawater and another was filled with seawater with ureadissolved at a concentration of 4 g tȂ1. When juveniles cultured in two different conditions for fiveweeks, the growth rate of the germlings of S. oligocystum cultured in seawater was always higherthan that of culture in seawater with urea dissolved. The results suggest that S. oligocystum has apotential to adapt to grow under lower nutrient environment.

Keywords : Sargassum oligocystum, morphogenesis, early development and growth, culture

1）Institute of Marine Science, Burapha University,Bangsaen, Chon Buri 20131, Thailand

2）Department of Aquatic Science, Faculty of Sci-ence, Burapha University, Bangsaen, Chon Buri20131, Thailand

3）Center of Excellence for the Oceans, NationalTaiwan Ocean University, 2 Pei-Ning Road,Keelung 20224, Taiwan

4）Faculty of Fisheries, Kasetsart University, Chatu-

jak, Bangkok 10900, Thailand5）School of Marine Biosciences, Kitasato University,Kitasato, Minami-ku, Sagamihara, Kanagawa, 228Ȃ8555, Japan＊Corresponding author: Thidarat NoiraksarTel: + 66(0)38 391671Fax: + 66(0)38 391674E-mail: sargassum2005@gmail.com

26 La mer 55, 2017

1. IntroductionSargassum C. Agardh is one of the largestgenus of brown algae and the most importantseaweed both ecologically and economically. TheSargassum plants are distributed all over theworld, especially in tropical and temperateregions（YOSHIDA , 1983). Seaweed beds consist-ing of Sargassum species influence the dissolvedoxygen content in seawater（hereafter, this isreferred to as DO）through photosynthesis（KOMATSU 1989; MURAOKA , 2004; MIKAM i et al.,2007）and consequently the pH value by CO2absorption through photosynthesis and releasethrough respiration（KOMATSU and KAWAI, 1986).They support biodiversity and habitat for marineorganisms（KOMATSU et al., 1982; KOMATSU, 1985;KOMATSU et al., 1990; KOMATSU and MURAKAMI,1994; KOMATSU et al., 1995; KOMATSU et al., 2007;KOMATSU et al., 2008). Sargassum species comprisebioactive compounds such as vitamins, carote-noids, dietary fibers, proteins, and minerals, andbiologically active compounds, like terpenoids,flavonoids, sterols, sulfated polysaccharides, poly-phenols, sargaquinoic acids, sargachromenol andpheophytin（LUCAS and SOUTHGATE, 2012). Sargas-sum species are used as human foods, especiallyby people living in coastal areas（e.g. KIRIMURA ,2007). There are many reports on the bioactivesubstances extracted from seaweeds, such asantibacterial, antifungal, antiviral, anti-inflamma-tory, anti-diabetic, antioxidant, and cytotoxicsubstances（e.g. ZANDI et al., 2010; TAJBAKHSH et al.,2011; YENDO et al., 2014; MEHDINEZHAD et al., 2015).Sargassum plants also play an effective bio-absorption role to remove nutrients（FEI , 2004）and heavy metals such as cadmium ion（Cd2+),copper ion（Cu2+), and mercuric ion（Hg2+）dis-solve in seawater. Therefore, this function ofSargassum species is focused from the environ-mental and economic aspects（RAMAVANDI et al.,2015; DELSHAB et al., 2016).

Many reports exist concerning the early de-velopment stages of Sargassum species such asS. micracanthum and S. ringgoldianum（OGAWA,1974) , S. muticum（NORTON , 1977; HALE s andFLETCHER , 1989; UCHIDA et al., 1991; KERRISON andLE, 2016), S. horneri（NANBA, 1993; UCHIDA, 1993;YOSHIDA et al., 1995; YOSHIDA et al., 1999; CHOI et al.,2008) , S. filicinum（YOSHIDA et al., 1999) , S.confusum（KAWAGOE et al., 2005), S. vachellianum（YAN and ZHANG, 2013), S. thunbergii（ZIGUO et al.,2008; YONGZHENG et al., 2015) , S. echinocarpum（HAMZA et al., 2016）and S. swartzii（KAVALE andVEERAGURUNATHAN , 2016) . In addition, there arereports on the technical development for artificialseed production in S. fulvellum（HWANG et al.,2006, 2007）and S. thunbergii（ZHANG et al., 2012).However, there is not any available informationon the embryo release and early development ofS. oligocystum which is one of the most commonand abundant species in tropical waters of thewestern Pacific Ocean.There are some extensive researches on fertil-izer application in seaweed cultivation（AMANOand NODA, 1987; BRAULT and QUÉGUINER, 1989;PHILLIPS and HURD, 2003; TYLER et al., 2005;MANSILLA et al., 2007; KIM and YARISH, 2014; MIKI etal., 2016). Urea is an organic compound with thechemical formula of CO(NH2)2 and is widely usedas a fertilizer for nitrogen source. Urea has thehighest nitrogen content of all solid nitrogenousfertilizers in common use and can get anywhereat a reasonable price. The standard crop-nutrientrating（NPK rating）of urea is 46Ȃ0Ȃ0, and it isalso used in many multi-component solid fertilizerformulations for land plants（WIKIPEDIA, 2016).However, it is unknown on the effect of urea onthe growth of S. oligocystum.Seaweed culture techniques have been devel-oped by researchers to observe the early devel-opment of seaweeds. Unfortunately, we have nodetailed studies on Thai Sargassum species until

27Morphogenesis and growth in the early life stages of Sargassum oligocystum

now. The objective of this study is to present themorphogenesis and growth in the early life stageof S. oligocystum from the fertilized egg stage tothe juvenile stage, and to test an effect of urea onthe growth of its juvenile plants. Materials werecultured under laboratory and outdoor condi-tions. Results were served for the objectives ofthis study.

2. Materials and methods2.1 Laboratory culture of fertilized eggsMature S. oligocystum plants were collected inthe intertidal zone of Samaesarn Island, ChonBuri Province, Thailand（12°31′21.37″N, 100°57′25.12″E）in April 2014（Fig. 1). The plants werecleaned to eliminate epiphytes and rinsed thor-oughly with sterilized seawater. Receptacleswere examined to check whether fertilized eggswere released and the eggs had attached to theirsurface or not. The fertilized eggs were removedfrom the receptacles by brush and rinsed severaltimes with sterile seawater. Plant Nutrition + liq-uid（Tropica, Aquacare）was used as a culturemedium and renewed once a week. Cultureconditions were as follows: a salinity of 30, a watertemperature of 25℃ and photosynthetic active

radiation（PAR）of 85 µmol photons mȂ2 sȂ1withthe use of cool daylight fluorescent tubes（Phi-lips, TLȂD 18W/54Ȃ765 1SL, Thailand）for a 12 h:12 h（L:D）（Figs. 2a, b). PARwas measured witha light meter（LIȂ250A, LIȂCOR, USA). Growthand development from the fertilized egg stage tothe juvenile stage for 90 d were observed. Juve-nile thalli cultured for 90 d were used for anoutdoor tank culture experiment.

2.2 Outdoor tank culture of juvenilesThree-month-old S. oligocystum juvenile thalliwere cultured in outdoor tanks of 500 L madefrom fiberglass, set under a roof with translucentplastic windows（Fig. 2c). To know the nutrition-al effects for the growth of S. oligocystumjuvenile, three hundred juvenile thalli werecultured in a tank filled with seawater and also atank filled with seawater with urea fertilizerdissolved at a concentration of 4 g tȂ1（hereafter,this is referred to as seawater with urea dissolvedfor simplicity）（Fig. 2d). The culture mediums inboth tanks were renewed once a week. Two repli-cates were used for each treatment. At intervalsof 7 d during 35 d of culture, fifteen young thalliwere randomly selected to measure the size ofjuvenile thalli under each treatment for examina-tion of their growth（Fig. 2e). In an outdoor tank,we measured eight environmental parameterssuch as water temperature, PAR（HOBO Pend-ant UAȂ002Ȃ64, USA), pH（Mettler Toledo pHFive Go, Switzerland), salinity（ATAGO 508 IIW,Japan).

2.3 Growth rate and data analysesA growth rate of a thallus was estimated froman increase in size of thallus. A specific growthrate for S. oligocystum was obtained with theformula proposed by Luhan and Sollesta（2010）:

Fig. 1 Mature thalli of Sargassum oligocystum ob-served around Samaesarn Island.

28 La mer 55, 2017

SGR= （W tȂW0) （1）

where SGR, t, W0 and Wt are specific growthrates, time of day after the start of outdoor tankculture, an initial size of thallus（mm）on the firstday of culture and a size of thallus（mm）at t,respectively. The first day t and a size Wt on the

first day of each week were set as 0 and W0because measurements were conducted at inter-vals of 7 d for 35 d. Differences in specific growthrates of S. oligocystum thallus per week wereexamined between those cultured in seawater orseawater with urea dissolved, and comparing theeight environmental parameters between the twodifferent mediums.

Fig. 2 Pictures showing laboratory cultures of Sargassum oligocystum germlings（a and b）;juveniles in outdoor tanks（c and d）in Samaesarn Island and the diameter measurementof the thallus（e).

29Morphogenesis and growth in the early life stages of Sargassum oligocystum

3. Results3.1 Field observation and embryo culture in a

laboratoryThe receptacle formation of S. oligocystum wasobserved from February to June around Samae-sarn Island, Chon Buri Province, Thailand. Fertil-

ized eggs released from conceptacles attached totheir surface. After verifying the start of germi-nation, zygotes were isolated in containers filledwith culture medium. The first segmentation inan egg occurred transversely to the longitudinalaxis of the egg and divided it into one large cell

Fig. 3 Embryo development of Sargassum oligocystum in a container filled with a culturemedium of Plant Nutrition+ liquid（Tropica, Aquacare）under a salinity of 30, a watertemperature of 25℃ and PAR of 85 µmol photons mȂ2 sȂ1. Pictures of fertilized eggs in areceptacle on the 1st day（a), a germling on the 3rd day with an arrow showing therhizoidal cell（b), a germling on the 7th day（c), a juvenile on the 30th day（d), a juvenileon the 60th day（e）and a juvenile on the 90th day（f).

30 La mer 55, 2017

and one small cell. The latter was graduallyinduced to rhizoidal cells after several divisionsand rhizoids grew out, and became a basal part ofthe germling for the attachment to substrate. Thecells from the former cell became an apex in 1 day

culture（Fig. 3a). Germlings produced many rhi-zoids in 3 day culture（Fig. 3b). They developedthe first cauline leaf in 7 day culture（Fig. 3c）andthe fourth cauline leaf came out in 30 day culture（Fig. 3d), becoming juvenile. The shape of theseleaves was lanceolate. Through the development,cauline leaves were lanceolate to spatulate in 60day culture（Fig. 3e), and broad-spatulate in 90day culture（Fig. 3f).In the seventh week of culture, juvenile thalli ofS. oligocystum developed a primary branch（Fig.4c). The average number of branches was 2.3Ȃ2.4with 8.3Ȃ10.9 mm in length from the 9th to 10thweek, 2.5Ȃ3.0 branches with 12.6Ȃ13.7 mm inlength between the 11th and 12th weeks, and2.9Ȃ3.5 branches with 13.0Ȃ13.3 mm between the13th and 14th weeks（Figs. 4d and 5a).

Fig. 4 Sargassum oligocystum juveniles in seawater（a）and seawater with urea dissolvedat a concentration of 4 g tȂ1（b）on the 5th day, with lateral branches in seawater on the7th day（c）in seawater on the 14th day, and（d）from the start of outdoor tank culture.

Fig. 5 Branch development of Sargassum oligocys-tum cultured in outdoor tanks from the 9th weekto the 14th week.

31Morphogenesis and growth in the early life stages of Sargassum oligocystum

3.2 Growth rate of juvenile cultured in outdoortanks

Three-month-old juveniles of S. oligocystum（hereafter referred to as young thalli）were usedfor a growth experiment cultured in tanks filledwith two different mediums: seawater or seawa-ter with urea dissolved. At the end of theexperiment on the 35th day, the average sizeof thalli obtained from the tanks filled with sea-water and seawater with urea dissolved were18.7±0.3 mm and 13.7±0.2 mm, respectively. Thehighest specific diameter of growth rates for thethallus in the former and latter tanks were2.7±0.2% dȂ1 on the 14th day and 1.3±0.2% dȂ1 onthe 7th day of culture, respectively（Fig. 6).Growth rate for the juvenile thalli of S. oligocys-tum cultured in the latter tanks were decreasedbecause of growth of blue-green algae on thethallus surface (Fig. 4b), while those in theformer tanks showed less growth of blue-greenalgae（Fig. 4a).The averages of environmental parameters areas follows: water temperature ranged from 30.4 to30.9℃; photosynthetic active radiation rangedfrom 21.7 to 40.5 µmol photons mȂ2sȂ1; salinity in

tanks filled with seawater and seawater withurea dissolved ranged from 32 to 33.9 and 31.5 to35.5, respectively; The pH in the former and lattertanks ranged from 8.1 to 8.3 and 8.2 to 8.4,respectively（Fig. 7). There was little differencein environmental parameters between the formerand latter tanks.

4. DiscussionThe germling development of S. oligocystum issimilar to that of tropical or temperate species ofSargassum such as S. confusum, S. horneri, S.thunbergii, S. swartzii and S. vachellianum（UCHIDA , 1993; KAWAGOE et al. , 2005; ZHAO et al,2008; YAN and ZHANG, 2013; KAVALE andVEERAGURUNATHAN, 2016). The development ofembryonic germlings in this species follows theclassic “8 nuclei in 1 egg” type, as described forSargassaceae. Fertilized eggs developed intoembryos at the primary-rhizoid stage in 24 h, andthe secondary-rhizoid stage in 3 d. The firstleaflet of the germling with cylindrical shape wasformed in 7 day culture. It is reported that cueson an egg release and the early germling growthof seaweeds were water temperature, irradiance,photoperiod, day length, nutrient, desiccation,thermal and osmotic stress（NORTON, 1977; UCHIDAet al., 2991; NANBA, and OKUDA, 1993; YOSHIDA et al.,1995, 1999; STEEN, 2004; STEEN and RUENESS, 2004;HWANG et al., 2006; CHOI et al., 2008; CHU et al., 2012;YONGZHENG et al., 2015). However, such cues couldnot be observed through our culture experiment.The specific growth rates of the juvenile thalliof S. oligocystum cultured in the tanks filled withseawater were higher than those cultured in thetanks filled with seawater with urea dissolved.This difference is due to the chemical compositionof the nutrient solutions used in this experiment.Urea is an excellent nitrogen source for someseaweeds such as kelps, but others show growthinhibition. For example, BRAULT and QUÉGUINER

Fig. 6 Growth rates of Sargassum oligocystumjuveniles（mean± standard error）at intervals of7 d, cultured in outdoor tanks filled with seawater（closed circle）and seawater with urea dissolvedat a concentration of 4 g tȂ1（closed square）for35 d.

32 La mer 55, 2017

（1989）studied the effect of inorganic and organicnitrogen sources on the growth of Ulva giganteanand found that ammonium was a better nitrogensource than urea and nitrate. PHILLIPS and HURD（2003）reported on nitrogen ecophysiology offour intertidal seaweeds（Stictosiphonia arbuscu-la, Apophlaea lyallii, Scytothamnus australis andXiphophora gladiata）from southeastern NewZealand and reported that there is a difference inabsorption by nitrogen sources and its seasonali-ty. The order of nitrogen sources well absorbedby seaweeds is NH4+> NO3Ȃ> urea in winter andNH4+= NO3Ȃ> urea in summer. MANSILLA et al.（2007）reported that Gigartina skottsbergii germ-lings grew more rapidly when they werecultured in solution of Bayfoland 250 SL andProvasoli than the growth rates cultured insolution with urea and superphosphate, whichwere significantly lower. Nitrogen and phospho-rus are limiting nutrients for growth and yield ofseaweeds in most natural environment. Physio-logical and biological factors of seaweeds mayhave an influence on growth and uptake ofnutrients, such as inter-seaweed variability, nu-tritional history, type of tissue, life history stage,age, surface area to volume ratio of a thallus, andmorphology.（HARRISON and HURD, 2001). HARRISONand HURD（2001）mentioned also that epiphytesgrowing on surfaces of seaweeds can controlseaweed growth to a critical level by starvingnitrogen uptake for several days. The presentstudy shows that the specific growth rates of S.oligocystum juveniles cultured in seawater withurea dissolved were decreased by blue-greenalgae contamination. It is possible that someattached algae may use the nutrients moreefficiently than S. oligocystum. It is estimated thatS. oligocystum succeeds to acquire a great abilityto adapt to the low nutrient level in tropicalwaters, especially in the waters of the east coastof the Gulf of Thailand.

AcknowledgmentsWe are deeply indebted to the sponsors of thisstudy which was conducted under the NationalScience and Technology Development Agency（NSTDA). Our thanks go to Plant Genetic Con-servation Project Under the Royal Initiation ofHer Royal Highness Princess Maha ChakriSirindhorn（RSPG）; Naval special warfare Com-mand, Royal Thai Navy; Institute of MarineScience and Faculty of Science Burapha Universi-ty; Atmosphere and Ocean Research Institute,The University of Tokyo; National Taiwan OceanUniversity; Faculty of Fisheries, Kasetsart Uni-versity; School of Marine Biosciences, KitasatoUniversity; the Asian CORE Program of theJapan Society for the Promotion of Science,“Establishment of research and education net-work on coastal marine science in Southeast Asia;and Core-to-Core Program of the Japan Societyfor the Promotion of Science, Research andEducation Network on coastal ecosystems inSoutheast Asia（RENSEA）for their supports.

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Received: December 12, 2016Accepted: January 17, 2017